Journal article
Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases
Methods in enzymology, Vol.592, pp.329-356
2017
DOI: 10.1016/bs.mie.2017.04.002
PMCID: PMC5772771
PMID: 28668126
Abstract
Translesion synthesis is the process by which nonclassical DNA polymerases bypass DNA damage during DNA replication. Cells possess a variety of nonclassical polymerases, each one is specific for incorporating nucleotides opposite to one or more closely related DNA lesions, called its cognate lesions. In this chapter, we discuss a variety of approaches for probing the catalytic activities and the protein-protein interactions of nonclassical polymerases. With respect to their catalytic activities, we discuss polymerase assays, steady-state kinetics, and presteady-state kinetics. With respect to their interactions, we discuss qualitative binding assays such as enzyme-linked immunosorbent assays and coimmunoprecipitation; quantitative binding assays such as isothermal titration calorimetry, surface plasmon resonance, and nuclear magnetic resonance spectroscopy; and single-molecule binding assays such as total internal reflection fluorescence microscopy. We focus on how nonclassical polymerases accommodate their cognate lesions during nucleotide incorporation and how the most appropriate nonclassical polymerase is selected for bypassing a given lesion.
Details
- Title: Subtitle
- Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases
- Creators
- Kyle T Powers - Carver College of Medicine, University of Iowa, Iowa City, IA, United StatesM Todd Washington - Carver College of Medicine, University of Iowa, Iowa City, IA, United States. Electronic address: todd-washington@uiowa.edu
- Resource Type
- Journal article
- Publication Details
- Methods in enzymology, Vol.592, pp.329-356
- DOI
- 10.1016/bs.mie.2017.04.002
- PMID
- 28668126
- PMCID
- PMC5772771
- NLM abbreviation
- Methods Enzymol
- ISSN
- 0076-6879
- eISSN
- 1557-7988
- Publisher
- United States
- Grant note
- R01 GM081433 / NIGMS NIH HHS P30 CA086862 / NCI NIH HHS
- Language
- English
- Date published
- 2017
- Academic Unit
- Radiation Oncology; Biochemistry and Molecular Biology
- Record Identifier
- 9984024541802771
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